Cellular localization and activity of Ad-delivered GFP-CFTR in airway epithelial and tracheal cells.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.

Adenovirus 5-fiber 35 chimeric vector mediates efficient apical correction of the cystic fibrosis transmembrane conductance regulator defect in cystic fibrosis primary airway epithelia.

In vivo gene transfer to the human respiratory tract by adenovirus serotype 5 (Ad5) vectors has revealed their limitations related to inefficient gene transfer, host antiviral response, and innate adenoviral toxicity. In the present work, we compared the cytotoxicity and efficiency of Ad5 and a chimeric Ad5F35 vector with respect to CFTR gene transfer to cystic fibrosis (CF) and non-CF human airway epithelial cells. We found that high doses of Ad5 vector had an adverse effect on the function of exogenous and endogenous CFTR. Results obtained with Ad5 capsid mutants suggested that the RGD motifs on the penton base capsomers were responsible for the negative effect on CFTR function. This negative interference did not result from a lower level of biosynthesis and/or altered cellular trafficking of the CFTR protein, but rather from an indirect mechanism of functional blockage of CFTR, related to the RGD integrin-mediated endocytic pathway of Ad5. No negative interference with CFTR was observed for Ad5F35, an Ad5-based vector pseudotyped with fibers from Ad35, a serotype that uses another cell entry pathway. In vitro, Ad5F35 vector expressing the GFP-tagged CFTR (Ad5F35-GFP-CFTR) showed a 30-fold higher efficiency of transduction and chloride channel correction in CFTR-deficient cells, compared with Ad5GFP-CFTR. Ex vivo, Ad5F35-GFP-CFTR had the capacity to transduce efficiently reconstituted airway epithelia from patients with CF (CF-HAE) via the apical surface, restored chloride channel function at relatively low vector doses, and showed relatively stable expression of GFP-CFTR for several weeks.

The coxsackie B virus and adenovirus receptor resides in a distinct membrane microdomain.

The coxsackie B virus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily. In addition to activity as a viral receptor, it may play a role in cellular adhesion. We asked what determines the cell membrane microdomain of CAR. We found that CAR is localized to a novel lipid-rich microdomain similar to that of the low-density lipoprotein receptor (LDLR) but distinct from that of a CAR variant that exhibited traditional lipid raft localization via fusion to a glycosylphosphatidylinositol (GPI) tail. The cytoplasmic tail determines its membrane localization, since deletion of this domain resulted in mislocalization. Results indicate that CAR, CAR-LDLR, and LDLR reside in a novel lipid raft that is distinct from caveolin-1-containing caveolae and GPI-linked proteins. Residence in a lipid-rich domain provides a mechanism that allows CAR to interact with other cell adhesion proteins and yet function as an adenovirus receptor.